Specific DNA-protein, DNA-nucleic acid, RNA-protein and RNA-nucleic acid interactions play important roles in the formation of stable structural components of cells, such as transcription complexes, ribosomes and small nuclear ribonucleoprotein particles. Additionally, these interactions are involved in many stages of gene expression and protein synthesis, playing both regulatory and catalytic roles in transcription, RNA processing, splicing, translation and protein targeting. Identification of the specific molecular interactions involved provides insight into the mechanisms of these processes and contributes to a better understanding of the general parameters of molecular recognition involving nucleic acids.
Photochemical cross-linking is a powerful tool for the identification of the specific interactions involved in DNA-protein and DNA-nucleic acid complexes. One approach involves incorporation of nucleotide analogs containing photoreactive cross-linking groups into the DNA. DNA-protein or DNA-nucleic acid complexes are then formed, the complexes are irradiated with ultraviolet (UV) light, and molecules covalently attached to the DNA by photo-cross-linking are identified. The use of such analogs results in much higher DNA cross-linking yields than direct cross-linking by excitation of unmodified nucleic acids. Nucleotide analogs conjugated to photochemical cross-linkers and placed at specific positions within a synthetic DNA molecule have been described in Yang S-W and Nash H. A., Proc. Natl. Acad. Sci. USA 1994, 91:12183-12187.
Nucleotide analogs that can be incorporated specifically into the 5'- and/or the 3'-end of RNA molecules also have been described. Such incorporations were achieved enzymatically by RNA polymerases, or chemically with some difficulty. Photo-cross-linking groups have also been placed at specific internal UMP residues in RNA utilizing automated chemical synthesis in a protected phosphoramide precursor. (Bradley, D., and Hanna, M. M., Tetrahedron Letters 1992, 33:6223-6226). These modified nucleotides, however, are somewhat labile and they have a very short range of cross-linking, often failing to make a sufficiently close contact with another micromolecule and reacting instead with water. Until recently, placement of photo-cross-linking analogs internally into RNA has been limited to uridine analogs. One of these analogs 5-((4-azidophenacyl)thio)-UTP, which contains a photoreactive azide group approximately 10 .ANG. from the base, is incorporated at internal positions in RNA by both E. coli and T7 RNA polymerases without interfering with normal Watson-Crick base pairing (Dissinger, S. and Hanna, M. M., J. Mol. Biol. 1991, 219:11-25). Other UDP analogs that are incorporated internally during transcription include 4-thio-UTP, 5-bromo-UTP, and 5 azido-UTP, all of which contain cross-linking groups directly on the nucleotide base and function essentially as 0 .ANG. probes (Woody, A-Y. M., et al., Biochem. Biophys. Res. Commun. 1988, 150:917-924).
Aryl azides are chemically inert until irradiated with long wavelength ultraviolet light, when a chemically reactive nitrene is generated on the azide. The nitrene can then insert rapidly and relatively nonspecifically into adjacent molecules, resulting in covalent attachment of the azide-tagged molecule to other molecules in the vicinity. This relatively non-specific insertion reaction makes azides excellent probes for the environment of a molecule, as one specific functional group need not be present on an adjacent molecule to the cross-linking. Using photo-cross-linking, proteins that specifically bind to nucleic acids can be identified, or conformational changes that occur in nucleic acid binding proteins upon interaction with other molecules can be detected.
In spite of the progress made in methodology, a number of problems have prevented the wide scale use of hybridization as a tool in human diagnostics. Among the more formidable problems are: i) the inefficiency of hybridization, and ii) the low concentration of specific target sequences in a mixture of genomic DNA.